Controlled expression of transgenes or of target endogenous genes will likely be essential for success of many genetic therapies. Constitutive expression of transgenes has resulted in down-regulation of effector systems and/or cellular toxicity in animal studies (Efrat et al, 1995, Proc. Natl. Acad. Sci. USA 92, 3576-3580). Regulated expression in response to metabolic, hormonal, or environmental signals is the normal situation for many eukaryotic genes. In order to mimic natural physiological expression patterns with transgenes, and to minimize interactions with human gene regulation signals, binary promoter/transactivator configurations of heterologous origin which respond to heterologous stimuli have been developed in recent years. However, many exogenous stimuli which modulate these artificial mammalian regulons have proven to be incompatible with human therapeutic use due to cytotoxicity or undesired side effects (Baim et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5072-5076; Braselmann et al., 1993, Proc. Natl. Acad. Sci. USA 90, 1657-1661; No D. et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351; Rivera et al., 1996, Nat. Medicine 2, 1028-1032; Suhr et al., 1998, Proc. Natl. Acad. Sci. USA 95, 7999-8004; Wang et al., 1994, Proc. Natl. Acad. Sci. USA 91, 8180-8184). The tetracycline-regulated mammalian expression system avoids these problems, and is described in U.S. Pat. Nos. 5,888,981; 5,866,755; 5,789,156; 5,654,168; and 5,650,298, to name just a few examples. However, the tetracycline-regulated system can fail to suppress gene expression adequately under repressed conditions.
Moreover, future gene therapy strategies will require technology which allows independent control of two different transgenes or sets of transgenes which are cotranscribed in a multicistronic configuration. For example, many tissue expansion and ex vivo gene therapy scenarios will require a two-step process beginning with expression of growth-promoting genes to enable expansion of grafted tissues in culture, followed by induction of growth suppressors to prevent tumorigenic behavior of treated cells after reimplantation. Sustained proliferation control is also required for stem cell-based technologies currently evaluated for eventual cell and tissue replacement therapy, since stem cells are tumorigenic (Rossant et al., Nat. Biotechnol. 17, 23-24; Solter et al., Science 283, 1468-1470). The second independent gene regulation system could be used in such cells for pharmacologic control of one or several secreted therapeutic proteins, such as insulin, to enable titration of circulating proteins into the therapeutic range or adapt expression to optimal daily dosing regimes. There is, therefore, a need for new mammalian gene regulation systems which employ modern, therapeutically proven antibiotics as controlling agents, and which can be used in combination with the tetracycline regulation system, with minimal interaction between tetracycline control and the new control modality.
The human oral antibiotic pristinamycin consists, like other streptogramins, of a mixture of two structurally dissimilar molecules, the streptogramin A component pristinamycin II (PII), a polyunsaturated macrolactone, and the streptogramin B component pristinamycin I (PI), a cyclic hexadepsipeptide. The water-soluble form of pristinamycin, Synercid, recently approved in the U.S. and Europe for use against most multiple drug-resistant Gram-positive bacteria (Barriuere et al., 1994, Expert Opin. Invest. Drugs 3, 115-131; Baquero et al., 1997, J. Antimicrob. Chemother. 39, 1-6), is composed of dalfopristin, a 26-sulphonyl derivative of PII, and quinupristin, which is derived from PI by synthetic addition of a (5.delta. R)-[(3S)-quinuclidinyl] thiomethyl group. Virginiamycin is another important streptogramin used as a growth promotant in livestock feed (Nagaraja et al., 1998, J. Anim. Sci. 76, 287-298). Either the A or B streptogramin components are individually bacteriostatic, but streptogramins A and B together are synergistically bactericidal (up to 100 times more active), a phenomenon which lowers incidence of acquired antibiotic resistance, since high level resistance to the combined streptogramins is likely only when both type A and type B streptogramin resistance determinants are present simultaneously (Cocito et al, 1997, J. Antimicrob. Chemother. 39, 7-13). Recently, a pristinamycin resistance determinant (ptr) has been cloned from S. pristinaespiralis (Blanc et al., 1995, Mol. Microbiol. 17, 989-999; Salah-Bey et al., 1995, Mol. Microbiol. 17, 1001-1012; Salah-Bey et al., 1995, Mol. Microbiol. 17, 1109-1119). Expression from the ptr promoter (containing a recognition sequence termed P.sub.PTR) is induced by pristinamycin, particularly by PI. A protein called Pip (pristinamycin-induced protein) was identified based on its affinity to P.sub.PTR in gel retardation experiments (Salah-Bey et al., 1995, Mol. Microbiol. 17, 1109-1119).
Recently, the prevalence of multidrug resistant human pathogenic bacteria has increased dramatically. This increase correlates with an escalation of bacterial disease and related mortality. Also, antibiotic chemotherapy is becoming more difficult as the percentage of elderly and immunocompromised patients grows. The European Commission has already reacted to this situation by banning the use of certain antibiotics as a growth promoter in livestock feed, among them the streptogramin virginiamycin, so as to limit the environmental spread of antibiotics (thought to be a major driving force for selection of multidrug resistant pathogenic bacteria) thereby preserving the use of antibiotics for human therapy.